The specific binding of a pair of peptides to each other triggers a vast number of functions in a living cell. For example, the specific binding of a ligand to a surface receptor serves as the trigger for cellular responses to many external signals. In mammals, cells respond to a wide variety of circulating peptide hormones, often through single transmembrane domain receptors. It is certainly recognized that the cytokine receptor superfamily illustrates the diverse aspects of cellular function, and physiological responses. Recent examinations of cytokine receptor function have revealed differing ligand-receptor protein stoichiometries including both 2-protein (ligand/receptor) (Cunningham et al., 1991; Staten et al., 1993) and 3-protein (ligand/receptor/receptor or ligand/receptor/transducer) interactions (Young, 1992; Taga and Kishimoto, 1992; Mui and Miyajima, 1994). The intricacies of such protein associations have been investigated using in vitro, often laborious, methods (Fuh et al., 1992; 1993; Davis et al., 1993) since cenetically malleable expression systems have been unavailable. The present invention is directed to novel modified host systems which can be used for such protein investigations, yet the novel systems are significantly less laborious.
Recently reported systems in the art refer to a "2-hybrid" system as discussed by Fields and Song, 1989 and also Chien et al., 1991. The "2-hybrid" sytem involves differential interactions between the separable DNA binding and activation domains of the yeast transcriptional activator, Gal4. Heterologous proteins are expressed as hybrid proteins fused to either half of Gal4 (see FIG. 1; Fields and Song, 1989; Chien et al., 1991 for discussion of the 2-hybrid system). The productive interaction of the heterologous proteins brings the two halves of the Gal4 protein in close proximity, activating expression of a scorable reporter gene. To this date, such 2-hybrid systems have been disclosed as useful for determining whether a first given test peptide sequence has binding activity for the known sequence of a second peptide; wherein the affinity of the test peptide for the known peptide is unknown. Studies using such a system have been directed to analyzing intracellular proteins such as transcription factors and kinase-target protein interactions (Yang et al., 1992; Durfee et al., 1993; Li et al., 1994).
The novel modified cells of this invention and novel methods incorporating these cells provide significant advancements for the study and discovery of peptide mimics, including ligand mimics and receptor mimics. At this time no one has developed an efficient and specific screening system to investigate these areas. By employing in the cell a peptide binding pair for which the binding affinity is known, the present invention permits the investigation of peptide binding pairs, such as a ligand and receptor, wherein the peptides bind via extracellular interactions. The present invention creates exponential advantages for the discovery or compounds which can interact as ligands for specific receptors or transducers. Potential ligands include, but are not limited to, mammalian hormones with the receptors being a cognate extracellular ligand-binding peptide. Furthermore, the present invention describes the use of cell systems which express multiple heterologous proteins, including the two heterologous fused proteins to establish the specific and reversible binding of the ligand and receptor. The specific interaction of the above-described binding is readily detected by a measurable change in cellular phenotype, e.g. growth on selective medium.